Cairo

MERS Found in African Camels

Middle East respiratory syndrome (MERS), the deadly viral disease discovered in Saudi Arabia in 2012, may be an African problem, too. Evidence suggests that camels in the Middle East are helping spread the disease, but researchers have now found it in camels shipped from Africa as well.

MERS has sickened 183 people and killed 80, most of them in Saudi Arabia. Malik Peiris, an infectious disease researcher at the University of Hong Kong, and colleagues collected samples from four Egyptian abattoirs and found that most camels carried antibodies against MERS in their blood. They also found MERS RNA—a sign of current infection—in nose swabs from four animals shipped in from Sudan and Ethiopia. Peiris cautions that camels could have picked up the virus on their final journey in Egypt, but says that "health authorities really need to test patients with severe pneumonia all across Africa for MERS." Another research team also reported last week that MERS is older than thought: Antibodies were present in camel serum samples from a Saudi archive going back to 1992. http://scim.ag/AfricaMERS

London

U.K. Proposes Regulations for 'Three-Parent Embryos'

The U.K. government last week issued draft regulations that would let researchers try a controversial new in vitro fertilization procedure in patients. The technique could allow women who are carriers of mitochondrial disease to have healthy, genetically related children by transferring the DNA from their egg cells into a donor egg cell that has healthy mitochondria. Such altering of genes of human egg cells or embryos is currently forbidden in the United Kingdom.

The proposal would permit the procedure only for women who are highly likely to pass on the disease. The mitochondrial donor would have no claim to parental rights, and donors and recipients would be kept anonymous, unless both parties wanted to meet. After a public comment period through 21 May, the Department of Health will present a final proposal to Parliament. The procedure is also under scrutiny in the United States, where advisers to the Food and Drug Administration last week expressed concerns that it is not quite ready for human clinical trials. http://scim.ag/mitoIVF

Tanegashima, Japan

Researchers Eye Tethers for Space Debris

The Japan Aerospace Exploration Agency (JAXA) on 28 February launched a satellite to test a scheme to rid space of zombie satellites and other junk orbiting Earth. The 9-kilogram Space Tethered Autonomous Robotic Satellite-2 (STARS-2) is the first step toward a new debris-gathering technique. Scientists hope to use space robots to connect a long conductive wire to each chunk of debris. Dragged through Earth's magnetic field, the tether generates an electric current that dissipates as heat, draining kinetic energy from the system. This electrodynamic drag should pull debris into the atmosphere, where it will burn up. Over the next 3 to 6 months, STARS-2 will test tether deployment and electricity generation as a step toward future missions that will actually capture debris.

Developed by researchers at Kagawa University in Takamatsu, STARS-2 was one of seven university-designed microsatellites piggybacking on the launch of the Global Precipitation Measurement Core Observatory, jointly developed by JAXA and NASA to monitor rain and snow worldwide.

Harbin, China

Infamous Experimentation Site to Become Historical Park

Amid rising political tensions with Japan and other Asian neighbors, China announced this week that it plans to construct a "cultural park" on the site of Unit 731, where scientists under the Imperial Japanese Army carried out horrific experiments during the Second Sino-Japanese War and World War II.

The army established the covert unit, officially called the Epidemic Prevention and Water Supply Department, in Harbin in 1936. For nearly a decade, scientists there infected men, women, and children with plague, anthrax, and other biological agents and conducted vivisection without anesthesia. Thousands of Russians, Chinese, and other Asians died in the experiments.

After World War II, the U.S. government agreed to conceal what happened if the researchers shared the results of the experiments. The new park will feature artifacts from biological warfare tests, but bioethicist Jing-Bao Nie of the University of Otago, Dunedin, in New Zealand suspects it could become "part of a larger place for entertainment and tourism, rather than one in memory of victims."

Barcelona, Spain

Devastated Agency Foresees Slow Recovery

Spain's largest public research organization faces years of eroded research activity and a decreasing workforce, its leadership revealed in a grim report released 26 February. The Spanish National Research Council (CSIC) action plan for 2014 to 2017 outlined efforts to address a lack of positions and limited management flexibility.

After seeing its government funding slashed by 36% from its 2008 peak, the council neared bankruptcy in 2013. It has lost more than 2000 staff members since 2011, most of them on temporary contracts, and government restrictions have prevented CSIC from filling research posts as scientists retire. The agency intends to start repairing the damage by resuming its Ph.D. and postdoc recruitment program, seeking government approval for a new tenure-track program, and helping scientists seek international grants.

Some Spanish scientists have commended the plan for being realistic, but critics worry that it fails to address the roots of the problem, including limited autonomy to pursue new research and a lack of strong leadership within CSIC institutes. http://scim.ag/_CSIC

Washington, D.C.

EPA Puts Hold on Copper Mine Permits

The U.S. Environmental Protection Agency (EPA) has halted the permit application for a large copper mine while it evaluates how to protect valuable habitat. The controversial Pebble Mine near Bristol Bay, Alaska, could produce as much as 300,000 metric tons of copper a year, but it threatens a fishery, worth $1.5 billion a year, which provides one-half of the world's sockeye salmon.

An EPA report found that the mine would destroy or bury 68 square kilometers of habitat and endanger other areas. Under the Clean Water Act, EPA lets the U.S. Army Corps of Engineers decide whether to issue a permit to harm wetlands. But the Pebble Mine is one of 30 cases where EPA has decided to evaluate the proposal itself.

Tom Collier, CEO of the Pebble Limited Partnership, called EPA's decision a "major overreach" based on inadequate research. The price of shares in Northern Dynasty Minerals, the sole remaining backer of the partnership, fell 30% after the decision was announced on 28 February.

Washington, D.C.

Obama Proposes Extra $5 Billion for Science, but Strings Attached

President Barack Obama presented a $3.9 trillion budget request to Congress on 4 March that includes about $135 billion for U.S. government research programs in the 2015 fiscal year, which begins 1 October. Overall, the proposed budget would give just small increases to the National Institutes of Health, the National Science Foundation (NSF ), the Department of Energy's Office of Science, and other heavyweight funding agencies. But it requests $5 billion in additional spending for a number of initiatives, including 1000 additional research grants at NSF; a new biosafety research laboratory in Athens, Georgia; and a new high-risk, high-reward funding program for biomedical science modeled on the military's Defense Advanced Research Projects Agency. Congress, however, is likely to balk at the tax and spending changes needed to free up that money. See Science's ongoing budget coverage at http://scim.ag/Budget15.

Random Sample

Noted

>Human genome sequencing pioneer J. Craig Venter has jumped with both feet into biomedical sequencing, this week announcing his latest venture, Human Longevity Inc. With $70 million in startup funding, the company plans to study cancer patients, and later, centenarians and children, by sequencing their genomes and cataloging their microbiomes. The goal is to harness these data to make long-term predictions about health and ultimately improve preventive medicine.

Epic Slide

A global network of seismometers has located the largest known natural landslide since 2010. On 16 February, a sheer face of Mount La Perouse in Glacier Bay National Park in Alaska collapsed. That day, while checking a list of seismic events, geophysicist Colin Stark of Columbia University's Lamont-Doherty Earth Observatory in Palisades, New York, noticed a signal indicative of a landslide. Analyzing the signal using a technique Stark helped develop (Science, 22 March 2013, p. 1416), colleague Göran Ekström and postdoc Clément Hibert reconstructed how the face collapsed, slammed into the top of the glacier, then slid over the ice. Perhaps 68 million tons of rock, snow, and ice hurtled 7.4 kilometers, they estimate. This satellite image, 10 kilometers across, was taken on 25 February.

Largest Rodent Could Be Lab Rat for Stroke Studies

Largest Rodent Could Be Lab Rat for Stroke Studies People undergo dramatic changes during puberty, but we've got nothing on the capybara. For reasons still mysterious to scientists, the world's largest rodent shuts off one of the main supplies of blood to its brain when it reaches sexual maturity. That makes the South American rodent an ideal natural model for studying stroke, a group of researchers in Brazil now proposes.

In both humans and capybaras, there are two main sources of blood to the brain: the internal carotid artery, which runs up each side of the neck, and the basilar artery, which begins at the base of the skull. When a capybara begins to sexually mature at about 6 months of age, its internal carotid artery becomes clogged with collagen and can no longer transmit blood. Still, "capybaras seem to cope very well" with the transformation, says University of São Paulo veterinary anatomist and surgeon Augusto Coppi. As the carotid artery closes, the rodent's basilar artery doubles in size to keep the brain supplied with blood.

When one of these arteries becomes blocked in a human, however, the other "doesn't have enough time to adapt," Coppi says—and the result is a stroke. In a recent paper in Cells Tissues Organs, he and colleagues argue that studying the capybara could help scientists figure out how to make human arteries behave more like their capybara counterparts and quickly pick up the slack if their companion shuts down.

Newsmakers

Three Q's

Since segueing from a Ph.D. in theoretical physics to filmmaking, Mark Levinson has done post-production work on movies including Cold Mountain and The Social Network. But Particle Fever—his first documentary—combines the two loves. Released this week, the film follows six physicists through the breakthroughs and heartbreaks leading to the discovery of the Higgs boson in 2012.

M.L.:In fiction films, usually you see stereotypes of scientists, and you don't really see the excitement, the humanity, what really drives them. That was something that I knew, and felt strongly about. … The idea was to do a narrative, dramatic story.

Q:Was it hard to generate that kind of excitement from 4 years of particle physics?

M.L.:The ingredients were there. We didn't have to generate that. The actual developments on their own ended up creating a narrative that we never could have dreamed of.

Q:How did the scientists respond to having their work filmed?

M.L.:They were really very open. I think they trusted the fact that we were going to show them accurately. The one requirement was, "Please, just don't make us look boring."

Structural Biology Scales Down

Robert F. Service

The United States is winding down a $1 billion project to churn out protein structures. What will that mean for the field?

The NDM-1 protein structure should help drugmakers fight this antibiotic killer.

CREDIT: V. ALTOUNIAN/SCIENCE

Three years ago, Andrzej Joachimiak decided to take on the superbugs. Infections from these antibiotic-resistant microbes are on an alarming rise globally, accounting for 2 million cases and 23,000 deaths a year in the United States alone. Among the most dangerous bugs are new strains with a protein known as NDM-1 that chops up a wide variety of previously effective antibiotics known as β-lactams, drugs that include penicillin.

Thanks to a long-running effort called the Protein Structure Initiative (PSI), Joachimiak had the tools to work out NDM-1's structure and pinpoint its weaknesses. Joachimiak, a structural biologist at Argonne National Laboratory in Illinois, and his colleagues used robots to synthesize 98 NDM-1 genes, each with subtle sequence variations. They succeeded in engineering bacteria to express 59 of those genes and produce their corresponding proteins at a high concentration. The researchers purified 53 of the proteins and coaxed 21 into forming crystals, many in combination with different druglike inhibitors and potential antibiotics. Then they shipped the best samples to the Advanced Photon Source, a stadium-sized synchrotron that fires a powerful beam of x-rays, bouncing them off crystalline solids to map their 3D atomic structures.

Joachimiak and his colleagues worked out 11 such atomic maps; others are still in progress. So far, the maps have shown that NDM-1 has an enlarged, flexible active site that allows it to fit, and ultimately break down, a wide variety of β-lactam antibiotics. Now, drug companies around the globe are free to use the results to design novel antibiotics that, someday, may save millions of lives. It was the PSI at its best, Joachimiak says.

The PSI cranked out protein structures and technologies. But rising costs squeezed competitive research grants.

CREDIT: H. BERMAN/RUTGERS UNIVERSITY

Since 2000, the U.S. National Institute of General Medical Sciences (NIGMS) has spent $907 million on the PSI, hoping to rev up the pace at which 3D protein structures like NDM-1 are solved; other institutes of the National Institutes of Health (NIH) chipped in another $23 million. That money funded large teams of biologists, physicists, chemists, and engineers to collaborate on not only determining protein structures, but also reinventing the way that this science is done. So far, PSI investigators have worked out the structures for 6507 proteins, 6.6% of all the structures with 3D data deposited in the international repository known as the Protein Data Bank (PDB).

But last fall, an NIGMS advisory council bowed to long-standing criticism of the PSI and pulled the plug on it, allowing its current round of funding to expire in June 2015. "In the current budget environment, in order to start a new program or bolster support for existing priorities such as investigator-initiated research, other programs must be adjusted or ended," NIGMS's new director, Jon Lorsch, wrote in a blog post in September 2013.

The announcement left longtime supporters of the PSI reeling and critics gleeful. But most of all, it has raised a string of questions: What was learned from the near $1 billion big-science experiment? What will happen to this team-oriented approach to biology? What will become of the high-speed facilities that were created? And what does the PSI's demise mean for the future of structural biology in the United States? "Structural biology really is at a crossroads," says Raymond Stevens, a structural biologist at the Scripps Research Institute in San Diego, California, and the leader of a PSI center devoted to solving structures of cell membrane proteins. "The PSI is dead. I view it as an opportunity to think about what's next."

The hunt is on

Before the PSI, structural biology was painfully slow. Typically, individual labs worked for months or years to clone a gene for a particular protein into bacteria or yeast cells and purify it. Then they often tried adding countless combinations of salts, buffers, and other additives to their protein-laced solutions to coax the proteins to arrange themselves into tiny crystals. The good ones could then be blasted with x-rays to see whether they would diffract in a tight pattern. After that, researchers often spent additional months or years mapping out the atoms. By the late 1990s, the PDB contained structures of only about 10,000 proteins. Meanwhile, the Human Genome Project was about to inundate researchers with genes for all the million-plus proteins in the human body. Determining their 3D structures would be a key step in sorting out their functions—and biologists realized that they would have to pick up the pace or fall hopelessly far behind.

Enter the PSI. In 2000, NIGMS officials laid out the program's goals. First, develop the technology needed to solve 5000 structures in 10 years. Then learn how to bypass crystallography altogether by using the solved structures to develop computer models that could take the gene sequence of an unknown protein and compute its likely 3D shape, giving insights into its function.

From September 2000 through June 2005, NIGMS spent $265 million on a pilot program, automating each phase of protein structure determination, including expressing proteins, purifying them, crystallizing them, collecting diffraction data at synchrotrons, and using software to solve their structures. More than 1100 structures later, NIGMS officials decided that the effort had succeeded well enough to push for a second "production" phase, PSI-2. From July 2005 through June 2010, NIGMS spent $346 million on four large-scale high-throughput centers, six specialized centers focused on developing methods for solving more challenging structures, and a pair of computer modeling centers. All told, the effort generated another 3700 structures. Most were unique, meaning that they shared less than 30% of their genetic sequence with any other protein and folded in ways no other protein did.

But the PSI also churned out controversy. The bulk of the newly discovered proteins came from bacteria, and researchers knew little about their function. PSI researchers argued that the bacterial proteins were teaching them basic rules of protein folding. But biologists outside the PSI wondered why so much effort was being spent pursuing proteins unlikely to improve human health. In 2007, a midterm review of the PSI-2's progress, led by University of Michigan, Ann Arbor, structural biologist Janet Smith, concluded that "the large PSI structure-determination centers are not cost-effective in terms of benefit to biomedical research." The reviewers recommended that the PSI be revamped to target proteins of high interest to biologists.

NIGMS obliged and funded a third phase of the program, dubbed PSI:Biology. Gone was the talk of seeking out unique ways in which proteins fold and obtaining structures of representatives of each protein "family." Instead, the four high-throughput centers and an additional nine centers refocused their efforts on solving biologically important structures.

Still, criticisms persisted. In a midterm evaluation of the PSI's third phase produced last year, yet another outside panel of biologists faulted the high-throughput centers. "[M]any of the projects being developed are technology driven, chosen because they can capitalize on the existing high-throughput structure pipelines, rather than being driven by biological interest or impact," the report stated. The panel recommended continuing PSI:Biology for another 3- or 5-year term beyond 2015. But it also advised NIGMS to begin thinking about how best to end the program and move structural biology away from a dedicated source of set-aside funding.

Disputed legacy

Lorsch and an NIGMS advisory panel jumped at the recommendation. They decided to forgo another phase and prepare right away for the transition, creating panels to work out what to do with the current PSI centers and all the equipment and technologies they have produced, and how best to fund structural biology going forward.

Opinions about NIGMS's decision are mixed. "The PSI was a bad idea from the start," says Stephen Harrison, a structural biologist at Harvard University and a longtime critic of the PSI. The initiative did speed technology development, he says, but much of that progress probably would have taken place anyway. Now that the program is being terminated, Harrison says, "structural biology can now go on where it should have gone all along": awarding grants to projects deemed most valuable by conventional peer review.

Joachimiak says such criticisms are too facile. According to one estimate, the cost of producing the structure for one of the easier "soluble" bacterial proteins has plunged about 56% since 2003 to about $50,000 per structure. A good chunk of the high-speed robotics and software that PSI labs developed for protein expression, purification, crystal growth, and x-ray structure determination are now in standard use by structural biology labs around the world. According to Helen Berman, an x-ray crystallographer at Rutgers University in Piscataway, New Jersey, who runs both the PDB and a PSI archive known as the Structural Biology Knowledgebase (SBKB), the PSI has produced 421 different technologies that have been either commercialized or disseminated through the SBKB online.

The PSI revolutionized a host of technologies, including robotic systems like this one for generating protein crystals en masse.

CREDIT: RIGAKU AUTOMATION

Beyond technology, Joachimiak and others argue that the PSI has made fundamental contributions to protein science. For example, Ian Wilson, a structural biologist at Scripps, and his colleagues have used the suite of tools at their high-throughput center to determine the structures of a large number of HIV and influenza viral proteins. Their goal is to identify common features in the proteins from each virus, which could provide targets for novel vaccines that would stop a wide variety of viral strains at once, rather than the one or two strains hit by current vaccines. And David Baker, a computational biologist at the University of Washington, Seattle, has used dozens of structures of stripped-down "ideal" proteins solved by the Northeast Structural Genomics Consortium—a PSI effort—to sort out rules for designing novel proteins never made by natural organisms. Baker and colleagues are now using those rules to design synthetic proteins to serve as gene therapy agents, catalysts for converting carbon dioxide into fuel, and a host of other applications.

But Michigan's Smith says projects such as Wilson's HIV work and Baker's protein design would have thrived anyway in a competitive funding environment of individual investigator awards, known as R01 grants. Meanwhile, she says, "there are a lot of problem-based structural biology projects of very high merit that are not getting funded right now, because there is not enough money." If NIGMS redirects some of the money now spent on the PSI into investigator-initiated grants, "this will be positive," she says.

Critics also fault the PSI for failing to identify enough rules of protein folding so that structures can be computed from their sequence, rather than laboriously solved. "There is no doubt that if you have a close [gene] sequence homology then you can do a lot of successful modeling," says Michael Levitt, a computational biologist at Stanford University in California. However, he adds, "protein folding has not yet been solved generally." PSI investigators concede the point. "Our computational methods still aren't strong enough yet," Stevens says. Levitt adds that even though PSI investigators have produced thousands of protein structures, the number of gene sequences encoding unknown proteins has grown much faster, to more than 30 million. As a result, Levitt says, "it would take a very long time and an enormous amount of money" to solve structures of representatives of a large percentage of protein families.

Such brute-force efforts are now off the table, and the current PSI centers will be dismantled over the coming years. "The question is, how can we make this transition as orderly as possible with minimal collateral damage?" says Smith, who serves on the panel of outside experts advising the PSI on how its assets should be distributed. One option is for NIGMS to continue to fund high-throughput protein expression, production, and crystallization facilities as centralized resources for the whole structural biology community to use. Another is to distribute some of these facilities and technologies among current structural biology labs. These high-speed tools "shouldn't just go away," Smith says. NIGMS hopes to decide between May and December of this year, after the panels are expected to submit their recommendations.

PSI investigators say dismantling their centers could imperil U.S. leadership in structural biology. "A lot of jobs will be ending," Stevens says. "We'll see a very significant drop-off in the number of protein structures coming from the U.S." Meanwhile, other countries, notably China, are ramping up their own efforts in high-speed structural biology. "I'm worried," Joachimiak says. "We've made incredible progress. Now we're looking at just shutting it down." Wilson agrees. "We need a balance" between R01-type work and larger scale projects, he says.

But Douglas Sheeley, a program officer at NIGMS who is overseeing the work of the two PSI transition panels, says the PSI's termination does not mean the agency is ending its support for structural biology or the collaborative team-based science that the PSI promoted. In 2012, NIGMS spent $164 million to support structural biology, roughly 70% of the NIH total.

That total will almost certainly go down, because it includes $75 million for the PSI. But Harrison insists that the U.S. structural biology community will thrive without the dedicated funds. NIGMS officials are considering using the PSI's budget to fund an increasing percentage of R01-type grants. Even if that money no longer supports structural biology, "I think that's okay," Harrison says. It will force all structural biology projects to justify their merit against all other research. "We should compete on an even playing field."

Dazzling History

Thomas Sumner

Over the past century, x-ray crystallography has transformed scientists' understanding of the structure and behavior of materials.

2013

Crystallography yields a detailed picture of the protein that HIV uses to invade immune cells.

Nobel Prize awarded for work

Gently Does It

Robert F. Service

A technique for crystallizing fragile biomolecules without disrupting them is helping researchers probe the structures of some of the body's most important but elusive proteins: those that usher other chemicals through the cell membrane.

Crystallizing proteins such as bacteriorhodopsin is key to solving their atomic structure.

CREDIT: VADIM CHEREZOV/THE SCRIPPS RESEARCH INSTITUTE

The tiny purple crystals, glistening within a translucent, fatty gel, signaled that Ehud Landau and Jürg Rosenbusch had made headway on one of the toughest problems in x-ray crystallography. To map a protein's atomic structure using x-rays, crystallographers have to coax its molecules to align themselves in crystals, like soldiers in perfect formation. That's difficult enough for ordinary proteins, which are complex, flexible molecules. But the membrane proteins that straddle the cell's surface and control the chemical traffic in and out are an even bigger challenge. Nestled within their normal protective environment, membrane proteins are stable and well-behaved. But take them out to try and get them to line up, and the task is like herding cats.

Two decades ago, Landau, a chemist then at the University of Basel in Switzerland, thought the answer might lie in a curious mixture of fatlike molecules called lipids, blended with water and other compounds. The concoctions spontaneously form 3D shapes called the lipidic cubic phase (LCP), and Landau hoped they could serve as a synthetic cell membrane to keep the membrane proteins happy outside cells. He and Rosenbusch, a structural biologist also at Basel, tested the scheme with a purple membrane protein known as bacteriorhodopsin (bR), found in halobacteria. The plan worked. The result was the 50-micron-wide bR crystals—and, in the years that followed, a mini-explosion in membrane protein crystal structures.

Membrane proteins may be the most important molecules in biology. These enzymes, receptors, channels, and transporters account for more than half of the targets for all pharmaceutical compounds on the market. And LCP has been essential for understanding them. "It's been magical for us," says Wayne Hendrickson, a protein crystallographer at Columbia University, who has recently used the technique to solve two membrane protein structures.

But getting the LCP mixtures right and handling them is tricky. After their first glimpse of those purple bR crystals, it took Landau and Rosenbusch several more years of tinkering before they could nail down the first high-resolution structure of the protein (Science, 12 September 1997, p. 1676). Now, however, thanks to decades of painstaking work by a small band of researchers, the technique is beginning to hit its stride.

Fits and starts

LCP wasn't the first technique that crystal growers used to enforce order among membrane proteins. Nor is it the most common approach even today. Both of those honors go to a technique that uses soaplike detergents to purify membrane proteins and get them out of cell membranes, a necessary step for getting them to crystallize.

Detergents contain two different kinds of compounds joined at the hip. On one end are hydrophilic groups, which readily associate with water. On the other end are fatty hydrocarbon chains. Dump the detergents into water in the right conditions and they form micelles, tiny spheres with the hydrophilic portion facing out into the water and the fatty hydrocarbon tails pointing inward to minimize their interaction with water. When lipid molecules, which have different hydrophilic groups linked to hydrocarbon tails, are added, the mix can form "bicelles" shaped like tiny disks made from a combination of the lipids and detergents, all with their hydrophilic portions facing out into the water.

Membrane proteins also typically contain one portion that prefers to associate with fatty membrane molecules, and two others that gravitate to the watery environment outside or inside the cell. So if you toss a membrane protein into a solution with micelles or bicelles, the water-fleeing portions of the protein will wedge themselves into the friendly confines of the hydrocarbons, stabilizing their structure. Add millions of copies of the same membrane protein, and if you're lucky they will all orient themselves the exact same way, making it possible for them to pack into an orderly crystal.

That strategy works in some cases. But often it goes spectacularly wrong. Sometimes the detergents are too harsh and rip apart the proteins. The tightly curved spherical micelles can wrench the proteins out of their normal shape, and subtle temperature differences can wreak havoc with bicelles.

Back in 1992, Landau thought LCPs might be a gentler option. LCPs have a gradually curving framework that arranges itself into a 3D grid surrounding a network of watery channels (see figure). Landau and Rosenbusch hoped the LCPs' combination of the lipid framework and watery channels would keep both parts of membrane proteins happy and the 3D grid arrangement might help orient them all in the same direction.

But LCPs "can be a hassle to work with," says Martin Caffrey, an LCP expert and membrane protein crystallographer at Trinity College Dublin. LCP is a clear goop with the consistency of toothpaste, Caffrey explains. While crystals can simply be filtered out of liquid detergent solutions, finding nearly invisible flecks of protein crystals inside the LCP is a real pain. The bR crystals were an exception: Their bright pinkish purple color made them stand out. "I was extremely excited," Landau says of the day in 1995 when he first spotted the tiny neon crystallites. "It was obvious to me that our concept had worked."

Of course, Landau and his colleagues still didn't have a structure. And their next problem was the x-ray beams produced by synchrotrons. These stadium-sized machines fire a staccato burst of densely packed x-rays at their targets. By tracking the way the x-rays diffract off their target, researchers can deduce the atomic structure of the material.

The trouble was that the LCP-grown bR crystals were significantly smaller than those produced in detergent micelles. Most synchrotron beams at the time were 100 microns across, or more—twice the width of the bR crystals. That meant that most of the x-rays in the beamline would whiz right by the bR crystallite and contribute nothing to the diffraction pattern.

Then fortune smiled: The newly built ESRF synchrotron in Grenoble, France, had just opened its first microfocus beamline for work on just such tiny crystals. Landau and Rosenbusch applied for time on the beam, got it, and quickly nailed down a crisp diffraction pattern for the protein. "This was an extraordinary breakthrough," Caffrey says.

CREDIT: V. ALTOUNIAN/SCIENCE BASED ON PROTEIN STRUCTURE FROM BRIAN KOBILKA

The question was whether the approach would work for other membrane proteins. Through the late 1990s, Rosenbusch, Landau, and others produced a string of successful x-ray structures with other colored membrane proteins, such as the greenish photosynthetic reaction center. "After that it got quiet," Caffrey says. Growth conditions that produce protein crystals in LCP invariably trap myriad tiny bubbles in the gel as well, making it even harder to pick out the crystals, if they were there at all.

Caffrey, then at Ohio State University, Columbus, set out to speed things up. In 2000, he and Vadim Cherezov, a postdoc, set about inventing new tools to speed the discovery of crystals in LCP. One was a "sandwich plate" that squished blobs of the clear goop between two glass plates, to make crystals easier to spot under a microscope. Another was a robot that automated the mixing of different lipids, salts, and buffers needed to crystallize each protein.

Despite a couple of years of rapid progress, LCP efforts nearly ground to a halt again in 2003 when Caffrey was recruited away from Ohio State to form a group dedicated to LCP and membrane protein crystallography at the University of Limerick, in his native Ireland. U.S. science funding agency rules stated that Caffrey was unable to take his robot and other equipment that had been paid for by U.S. taxpayers. Cherezov faced an uncertain future as well. But things took a welcome turn when Cherezov went to San Diego, California, to visit a friend who worked at the Scripps Research Institute. There he met Raymond Stevens, a renowned structural biologist. After inviting Cherezov to give a seminar on LCP, Stevens asked him to join his group.

"Something that no one has ever seen"

The new landing spot was an ideal fit. At the time, Stevens was collaborating with Brian Kobilka, a biochemist at Stanford University, on attempts to crystallize membrane proteins known as G
protein–coupled receptors (GPCRs). GPCRs are one of medicine's most important sets of membrane proteins, as they transfer chemical signals from outside cells to G proteins inside cells. The G proteins, in turn, launch a variety of molecular dominoes that govern everything from your heart rate to your sense of smell.

By the mid-2000s, Kobilka had managed to grow crystals of a GPCR known as the β2 adrenergic receptor (β2-AR)—a cell-signaling component involved in everything from heart muscle contraction to digestion—in conventional lipid micelles. But the crystals were poor and didn't diffract well, Kobilka says. Like many other membrane proteins, β2-AR is a Janus molecule. The part that prefers to nestle within the fatty membrane usually keeps an orderly and stable structure. But the section that protrudes into the watery surroundings flops around like a flag in the wind. Kobilka's lab was struggling to find ways to stabilize those floppy portions to ensure that all copies of the protein lined up in the same manner inside a crystal. The researchers got partway there by adding copies of an antibody that grabbed part of the floppy portion of the β2-AR and held it in place. Then they grew the protein-antibody complexes in bicelles. The result, published in Nature, was one of the first crystal structures of a GPCR. The crystal difracted to 3.4 angstroms, a resolution that reveals most of the protein's amino acids.

In hopes of seeing even more detail, Kobilka and colleagues tried another tack. They clipped off a particularly unwieldy portion of β2-AR and replaced it with an orderly protein called T4 lysozyme, and grew those hybrids in bicelles. This got them crystals that diffracted to 4.2 angstroms. So they sent a batch of these hybrid membrane proteins to Stevens's lab. After a few months spent optimizing the LCP conditions, Cherezov produced high-quality crystals, and the researchers took them to the microfocus beamline at the Advanced Photon Source at Argonne National Laboratory in Illinois. The result was a 2.4 angstrom resolution structure (Science, 23 November 2007, p. 1258), which Science named one of its top 10 breakthroughs of the year.

Next, Kobilka wanted to see if he could get the structure of a GPCR bound to its G protein mate, which would show the GPCR's conformation in its "on" state. But Stevens, and his postdoc Cherezov, wanted to explore the broader landscape of GPCRs; humans alone have an estimated 800 varieties. So Kobilka teamed up with Cherezov's former mentor, Caffrey. The G protein turned out to be a behemoth, roughly twice as big as the GPCR. That made it too big to fit into the 50-angstrom-wide watery channels in the LCP. Kobilka hoped to find a way to make the channels bigger.

Back when Caffrey was at Ohio State, he had experimented with dozens of different lipids, charting their effect on the shape and size of the LCP network. He told Kobilka he thought they could widen the channels by replacing the conventional lipid in LCP, known as monoolein, with a shorter chain lipid known as 7.7 MAG.

Caffrey was right. In 2011, using 7.7 MAG for their LCP, along with other changes, Caffrey, Kobilka, and their colleagues were able to get crystals of the complex and work out the structure. "There have been three to four times in my career where I have seen something that no one has ever seen before. It was very exciting," Kobilka says. Caffrey agrees. "It was an extraordinary achievement," he says of Kobilka's structure of the complex, which helped earn Kobilka a share of the 2012 Nobel Prize in chemistry. "The cubic phase was just part of it, but an important part."

Membrane proteins control the chemical traffic into and out of cells and account for more than half of all drug targets.

LCP's success has been equally important for Stevens. In collaboration with Cherezov, who has since moved into his own faculty position at Scripps, Stevens's lab has now solved 16 of the 24 GPCR structures completed to date. The collection now represents four of the five major families of GPCRs.

Stevens, Cherezov, Caffrey, and others recently made another leap forward when they adapted a beamline at the free-electron laser (FEL) at the Center for Free-Electron Laser Science in Hamburg, Germany, to solve structures of LCP-derived crystals of membrane proteins with unprecedented efficiency (Science, 20 December 2013, p. 1521). FELs represent the latest in synchrotron technology, able to produce x-ray beams that are tighter and pack more than 1 billion times more photons into a given area than ever before. The beams are so powerful, in fact, that they vaporize crystals as soon as they hit them. But because the x-ray photons are traveling at the speed of light, they still manage to diffract well before the slow-moving atoms in the crystal explode outward.

The trick is zapping enough crystals to build up sufficient data to solve a protein's structure. In 2011, researchers led by Henry Chapman at the Center for Free-Electron Laser Science and Petra Fromme and Uwe Weierstall at Arizona State University, Tempe, had designed a device for injecting detergent laden with membrane protein crystals into an FEL beamline and showed the setup produced enough diffraction data for the team to solve the structure of an abundant membrane protein. But the technique was a huge waste of crystals. FEL beamlines don't shine a continuous beam of x-rays. Rather, they send them in dense packets 120 times a second. In between those bursts is essentially dead space that produces no data. To ensure that the x-ray bursts would hit enough crystals, Chapman's team had to spray in a steady stream of the detergent-and-crystal mixture. The x-ray packets hit only about one crystal in 10,000; the others produced no data. "It's hugely wasteful" and thus can't be used with most membrane proteins, which can be harvested only in tiny amounts, Caffrey says.

The LCP aficionados asked Fromme and her injection-builder colleagues to remake their injector to work with the LCP gel. A redesign worked. When the thick LCP goop is pushed through a tiny injector nozzle, it forms a continuous "stream" at a much lower velocity than the previous liquid stream, much as toothpaste emerges from a tube more slowly than a jet of water from a hose. The result was that far more crystals were hit by x-ray packets and the crystal losses were reduced between 100- and 1000-fold.

That triumph should help LCP's successes continue to roll in. Cherezov notes that in the past 2 years, structural biologists have solved more than 25 unique membrane protein structures with LCP—more than in all previous years combined. LCP-aided structures now account for 25% of all solved membrane structures, a fraction that is growing rapidly.

That doesn't mean the membrane protein crystallography challenge has been solved. "LCP is not a panacea," as it still doesn't work with some of the larger protein complexes, Cherezov cautions. But clearly, Stevens says, the logjam has broken. "For single membrane proteins, for the most part, if we want to get a structure we can get it," he says. With drugmakers now turning to membrane protein structures to identify novel targets for new classes of drugs against everything from pain and depression to heart disease and migraine headaches, LCP's success may soon make a difference in millions of peoples' lives.